Cardiac hypertrophy is initially a compensatory response that augments cardiac output. However, sustained hypertrophy leads to dilated cardiomyopathy and heart failure (HF), which kills more than 262,000 lives a year in United States. Recent studies have demonstrated that the pathological increase of intracellular calcium [Ca2+]i is the primary cause of triggering cardiac hypertrophic gene program. Interestingly, blocking or genetic deletion of transient outward K+ channel (Ito) is capable of triggering cell growth and cardiac hypertrophy. Taken together, Ito may participate functional regulation of [Ca2+]i in cardiac myocytes. We recently reported that blocking of Ito significantly facilitates L-type calcium channel current (ICa) in mouse ventricular myocytes and we hypothesize that there is a functional association between Ito and ICa and that the Ca2+/Calmodulin- dependent kinase II (CaMKII) is involved in the transduction of regulatory signals between Ito and ICa. Here, we extend our work to test these hypotheses. Our first aim is to test whether Ito channel and CaMKII form a macromolecular complex by using techniques of co-immunoprecipitation and immunocytochemistry labeling. The second aim is to test whether CaMKII- Ito channel complex is dynamically functioning in response to Ito blocking (i.e. whether CaMKII will be displaced by Ito channel blocker). The third aim is to test whether ICa facilitation is the result of Ca2+ channel phosphorylation by CaMKII that displaced from CaMKII-Ito channel complex by Ito channel blocker. The electrophysiological (whole-cell patch clamp) and genetic (CaMKII d knockout mouse model) approaches will be employed to test these hypotheses. The proposed concept here is that Ito channel is a reservoir for CaMKII and the displacement of CaMKII by Ito channel blocker increases CaMKII available to facilitate ICa. Although high risk is anticipated due to the early and conceptual stage, the feasibility is strongly suggested by our recently published electrophysiology results and the co-immunoprecipitation data from other group. Testing the proposed hypothesis may lead to a significant and new conceptual framework and a breakthrough in a new area of research. For instance, the role of functional association of ICa and Ito in the development of cardiac hypertrophy and failure will be the immediate area of interest. Cardiac hypertrophy and the consequent heart failure kill more than 262,000 lives a year in United States. This exploratory proposal is focused on a membrane K+ current (transient outward K+ current, Ito) which is consistently found to be decreased in heart failure as a secondary change. Our overall goal is to test whether blocking this membrane ion channel facilitates Ca2+ influx of cardiomyocytes via functional regulation of the cardiac Ca2+ channel (L-type Ca2+ channel) and to explore the underlying mechanisms which may lead to a significant and new conceptual framework of which the down-regulation of Ito may play an important role in promoting the progression of HF process. [unreadable] [unreadable] [unreadable] [unreadable] [unreadable]